The present exemplary embodiments relate to monitoring reactivity in biological and non-biological reactions. It finds particular application in conjunction with the reactivity between materials in an array, such as a microarray or array of wells of a microtitre plate, and will be described with particular reference thereto. However, it is to be appreciated that the present exemplary embodiments are also suitable for use in monitoring in non-array environments as well.
Researchers are increasingly employing combinatorial chemistry techniques in a variety of areas. In the pharmaceutical-industry, the testing of new candidate molecules for binding to a protein, nucleic acid, or other macromolecules of interest is an active area of research with numerous and diverse applications. In addition, there is a great interest in developing new antibodies to catalyze the formation of novel compounds, to catalyze the degradation of unwanted compounds, to modify biological pathways, and to act as therapeutic agents for drug overdose, biological warfare agent exposure, and other conditions caused by particularly potent antigens and poisons.
To test the reactivity in both binding and catalytic reactions of these molecules, researchers are using various techniques, including microarrays and “lab-on-a-chip” type devices. In such techniques, researchers can rely on fluorescent tags to test for reactions between subject molecules. While effective, fluorescent tags must be attached to each candidate compound prior to testing. This process is cumbersome and makes the testing of large numbers of samples time consuming. The article “Catalytic Antibodies: Structure and Function”, P. Wentworth and K. Janda (Cell Biochemistry and Biophysics, vol. 35, pp. 63-87, 2001) illustrates many of the problems faced, and gives examples of procedures followed by researchers employing combinatorial chemistry techniques to develop new antibodies.
One technique that researchers are using for real-time, high-throughput monitoring of fast reactions relies on nanocalorimetry-based processes, such as described in commonly assigned U.S. patent application Ser. No. 10/114,611 filed on Apr. 1, 2002 and titled “Apparatus and Method for a Nanocalorimeter for Detecting Chemical Reactions”, hereby incorporated by reference. While effective for samples where the reaction produces sufficient heat in a time scale normally on the order of up to several to tens of seconds, nanocalorimetry is not suitable in applications where the reaction is too slow or too weak to produce a detectable heat. This problem is especially pronounced for those reactions which may require minutes or even hours.
In investing alternatives, it has been appreciated that osmotic pressure may provide a useful measurement. Particularly, it is known that the osmotic pressure of a solution is a colligative property that depends on the concentration of solute molecules in the solution. For dilute solutions, the osmotic pressure Π obeys the equation Π=cRT, where c is molar concentration of solute, R is the gas constant, and T is the absolute temperature. Essentially, each mole of solute contributes RT thermal energy to the osmotic pressure.
In a biological test for reaction between a first material and second material of interest, e.g., a protein, and a candidate “probe” compound (or ligand), consider the case where both species are initially present in the same molar concentration, N, in a reaction cell. The use of the terms “first material” and “second material” may be used interchangeably herein with the terms “material 1” and “material 2”, respectively, and are intended to be synonymous unless specifically stated. In this initial, unreacted state, the combined concentration of both species is 2N, and each species contributes equally to the osmotic pressure in the cell. If the first material reacts with the second material to form a bound complex molecule, then N moles per unit volume of the first material reacts with N moles per unit volume of the second material to produce N moles per unit volume material 1-material 2 complex. Accordingly, the osmotic pressure due to these two components drops to ½ its previous level prior to binding.
Conversely, if the reaction of interest is catalytic in nature, for example in the case of a catalytic antibody reaction with an antigen in which the catalytic antibody cleaves the antigen, then N moles per unit volume of material 1 react with N moles per unit volume of material 2 to form 2N moles per unit volume material 2 fragments plus the original N moles per unit volume of material 1. In this case, the osmotic pressure increases by ½. The osmotic pressure is also a parameter which may be monitored over an extended period of time, such as, for example, for up to several hours.
There have therefore been attempts to use osmotic pressure to test for reactivity in both biological systems. However, these systems have generally examined high concentration environments in which the osmotic pressure is no less than approximately 5,000 to 10,000 N/m2. Furthermore, previous systems typically tested for only a single reaction at a time.
In many cases, however, it is desirable to undertake studies at low concentrations, which will generate osmotic pressures at levels much lower than present osmotic-based systems are capable of detecting. One reason the use of low concentrations is attractive, is that the materials may be scarce and/or expensive, making use of larger concentrations impossible or cost prohibitive. Additionally, the quantity of experiments may require the use of low concentrations. In drug screening experiments, for example, researchers may be running anywhere from 1,000 to 100,000 or more different experiments. The use of large concentrations of materials would significantly increase the cost to such a large number of experiments.
Another benefit of low concentration studies is that the use of smaller concentrations provides for more selective reactions. Consider, for example, the study of a binding reaction with a dissociation constant Kd:
            A      +      B        →          C      ⁢                          ⁢              K        d              =                    [        A        ]            ⁡              [        B        ]                    [      C      ]      In this reaction, A and B bind to form the complex C, and the dissociation constant is written in terms of concentrations denoted by square brackets. This equation assumes ideal solution behavior, but it is sufficient for the purposes herein. In testing for binding, it is often desired to obtain an indication of the magnitude of Kd. In many biochemical studies, including drug screening and development studies and proteome-wide investigations of protein-protein interactions, among others, Kd values of interest are typically <1-10 μM, and values from 1-1000 nM—and especially <100 nM—are not uncommon and often of particular interest. In order to measure Kd, the reaction must be studied at concentrations that are not too distant from the value of Kd. At the upper end of this range, titrations may be performed at concentrations of 10 to 100 times Kd, but titrations at concentrations near the value of Kd are preferred when possible. Thus, there is a benefit to performing studies at as low a concentration as possible. In particular, there is a benefit to being able to perform studies at concentrations as low as 10−6 to 10−7M. Likewise, it is a benefit to be able to measure kinetics of enzymatic reactions at low concentrations, including enzymatic reactions with slow turnover rates.
Upon a review of the state of art, it has been determined that there are no direct, simple, and generic assay techniques or systems for testing large numbers of samples of interest at the low concentration levels of interest. Available techniques require tagged molecules (e.g. for fluorescent, calorimetric, spectrophotometric, or radiolabelled tags), immobilization of reactants at or near a surface, antibody-based affinity screens, or other specific preparations that either modify the reacting molecules or are otherwise specific to the particular compounds being tested.